Pharmaceutical Formulation

ABSTRACT

The invention relates to aqueous pharmaceutical formulations comprising human growth hormone, histidine, poloxamer, phenol, and mannitol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/349,480, filed on Jan. 22, 2003 (published as US-2003-0162711 -A1),which is a continuation of U.S. application Ser. No. 08/842,293, filedon Apr. 23, 1997, and claims priority under 35 U.S.C. 119 of Danishapplication no. 0490/96 filed on Apr. 24, 1996, and U.S. provisionalapplication No. 60/028,620, filed on Aug. 28, 1996, the contents ofwhich are fully incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to novel pharmaceutical formulations comprisinggrowth hormone, or a derivative of growth hormone, and an amino acidselected from the group of Asp, Ile, Val, Leu, or His, or a derivativeof histidine, or a peptide comprising at least one basic amino acid andat least one acidic amino acid, and a non-ionic detergent, e.g.polysorbate or poloxamer. The invention further relates to a method ofproducing such formulations, and a method of treatment using suchformulations.

BACKGROUND OF THE INVENTION

The growth hormones from man and from the common domestic animals areproteins of approximately 191 amino acids, synthesized and secreted fromthe anterior lope of the pituitary gland. Human growth hormone consistsof 191 amino acids.

Growth hormone is a key hormone involved in the regulation of not onlysomatic growth, but also in the regulation of metabolism of proteins,carbohydrates and lipids. The major effect of growth hormone is topromote growth.

The organ systems affected by growth hormone include the skeleton,connective tissue, muscles, and viscera such as liver, intestine, andkidneys.

Until the development of the recombinant technology and the cloning ofthe growth hormone gene now giving rise to production of e.g. humangrowth hormone (hGH) and Met-hGH in industrial scale, human growthhormone could only be obtained by extraction from the pituitary glandsof human cadavers. The very limited supplies of growth hormonerestricted the use thereof to longitudinal growth promotion in childhoodand puberty for treatment of dwarfism, even though it has been proposedfor inter alia treatment of short stature (due to growth hormonedeficiency, normal short stature and Turner syndrome), growth hormonedeficiency in adults, infertility, treatment of burns, wound healing,dystrophy, bone knitting, osteoporosis, diffuse gastric bleeding, andpseudoarthrosis.

Furthermore, growth hormone has been proposed for increasing the rate ofgrowth of domestic animals or for decreasing the proportion of fat.

Pharmaceutical preparations of growth hormone tend to be unstable.Degradation products such as deamidated or sulfoxydated products anddimer or polymer forms are generated—especially in solutions of growthhormone.

The predominant chemical degradation reactions of hGH are 1) deamidationby direct hydrolysis or via a cyclic succinimide intermediate to formvarious amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH, andD-iso-asp-hGH (ref 1-3), 2) oxidation of the methionine residues inpositions 14 and 125 (ref 4-9), and 3) cleavage of peptide bonds.

Deamidation especially takes place at the Asn in position 149.

hGH is rather easily oxidized in positions 14 and 125, especially insolution (4-8), since the oxidation of hGH in solution formingsulfoxides is normally due to the oxygen dissolved in the preparation.

At present, it is not believed that these degradation products shouldhave toxic or altered biological activity or receptor bindingproperties, but there is indication to the effect that the conformationstability of the sulfoxides is reduced as compared to native hGH.

Other “degradation products” of growth hormone are aggregation productssuch as dimers and polymers. Studies have been performed to clarify therole of these forms in inducing an immune response with measurableamounts of antibodies to native hGH. These studies have indicated thataggregates of hGH are the primary cause of immunogenicity in patients.

Thus, it is desirable in a pharmaceutical formulation to avoid theformation of aggregates of growth hormone since such aggregates arecapable of causing undesirable immunogenicity or altered half-life.

The kinetics of degradation depend on temperature, pH and variousadditives or adjuvants in the hGH formulation.

Due to the instability, a growth hormone formulation is at presentnormally lyophilized and stored in the lyophilized form at 2-8° C. untilit is reconstituted for use in order to minimize the degradation. Thelyophilized pharmaceutical preparations comprising hGH are reconstitutedby the patient and then stored as a solution during the use for a periodof up to 30 days, during which some degradation will take place.

It is at present preferred to reconstitute the growth hormone as late aspossible before use and to store and ship the preparation in alyophilized state. The chain from the manufacturer to the pharmacy isapt for handling the preparations at a controlled low temperature ofe.g. 2-8° C. which allows for a shelf life of up to three years.

Preferably, a lyophilized and then reconstituted preparation should bestable with the end user in a lyophilized state for about one month andadditionally for one month in a reconstituted state in a pen device forthe intended period of use of a cartridge.

As an alternative to lyophilized and reconstituted preparations, growthhormone may be formulated as a liquid formulation suitable for use invials or in a pen system for self-medication. The extended use of pensystems for self-medication and the expanded field of use calls for apreparation which is stable for a sufficient long time with the enduser. Such stabilization is of very great importance when moving theadministration of the growth hormone from clinics to the homes of theindividuals to be treated where optimal storage conditions may not beavailable. A “ready to use” formulation furthermore diminishes anyhandling problems in connection with reconstitution and thus representsa convenience for the patient.

A stable dissolved preparation comprising growth hormone may be producedready to use in the form of vials used by the patient in combinationwith conventional syringes or as cartridges fitting into the pen deviceused by the patient. In both cases, the patient may then avoid thereconstitution of the preparation and, hence, will not have to be in thepossession of a lyophilized preparation, a suitable vehicle forreconstitution as well as the necessary skill and sterile equipment forsterile reconstitution of the preparation. Safety reasons also make itdesirable to avoid the reconstitution of a lyophilized preparation justbefore the use of the preparation.

From the manufacturer's point of view, it may be an advantage to avoidthe lyophilization step in the production of growth hormonepreparations. Lyophilization is a time consuming and costly process andis also often a “bottleneck” in the production due to the limitedcapacity of the freeze drier.

Thus, there is a need for more stable dissolved preparations of growthhormone in order to facilitate the handling to be performed by thepatient.

Thus, there is also a need to reduce the rate of the degradationprocesses in order to allow for dissolved hGH preparations being stableduring shelf life and during the period of use of up to about one month.

Prior art

Prior attempts to stabilize hGH has not fully succeeded in preventingthe formation of dimer. The problems associated with dimer formation ise.g noted in Becker, G. W., Biotechnology and Applied Biochemistry 9,478 (1987).

International Patent Publication No. WO 89/09614 and Australian patentapplication No. 30771/89 disclose a stable pharmaceutical formulationcontaining human growth hormone, glycine, and mannitol. Such aformulation shows improved stability during normal processing andstorage in a lyophilized state as well as in the period of use after thereconstitution.

Published European patent application No. 303 746 discloses that animalgrowth hormone may be stabilized with various stabilizers to givedecreased formation of insolubles and preservation of the solubleactivity in aqueous environments, such stabilizers including certainpolyols, amino acids, polymers of amino acids having a charged sidegroup at physiological pH, and choline salts. Polyols are selected fromthe group consisting of non-reducing sugars, sugar alcohols, sugaracids, pentaerythritol, lactose, water-soluble dextrans and Ficoll;amino acids are selected from the group consisting of glycine,sarcosine, lysine or salts thereof, serine, arginine or salts thereof,betaine, N,N,-dimethyl-glycine, aspartic acid or salts thereof, glutamicacid or salts thereof, a polymer of an amino acid having a charged sidegroup at physiological pH may be selected from polylysine, polyasparticacid, polyglutamic acid, polyarginine, polyhistidine, polyornithine andsalts thereof, and choline derivatives are selected from the groupconsisting of choline chloride, choline dihydrogen citrate, cholinebitartrate, choline bicarbonate, tricholine citrate, choline ascorbate,choline borate, choline gluconate, choline phosphate,di(choline)sulphate and dicholine mucate.

U.S. patent specification Ser. No. 4,917,685 discloses a delivery devicedesigned to be implanted comprising growth hormone stabilized using thesame stabilizers as mentioned in EP 303746.

Published European patent application No. 374,120 discloses a stabilizedformulation comprising hGH and a polyol having three hydroxy groups.Glycerol and tris(hydroxymethyl)aminomethane are mentioned. Furthermore,the presence of histidine hydrochloride as a buffer together with thepolyol is disclosed.

International Patent Publication No. WO 93/12811 discloses stabilizedformulations of growth hormone in the form of a lyophilized powder or anaqueous solution comprising asparagine.

International Patent Publication No. WO 93/12812 discloses stabilizedformulations of growth hormone in the form of a lyophilized powder or anaqueous solution comprising histidine. In such formulations thedeamidation is reduced by 25-30% as compared to a correspondingformulation of growth hormone comprising phosphate buffer.

International patent Publication No. WO 96/11703 discloses stabilizedformulations of growth hormone comprising isoleucine.

International patent Publication No. WO 96/11702 discloses stabilizedformulations of growth hormone comprising valine. International patentPublication No. WO 96/11704 discloses stabilized formulations of growthhormone comprising leucine.

International Patent Publication No. WO 93/19776 discloses proteinformulations comprising growth hormone comprising citrate as buffersubstance being more stable than formulations comprising phosphatebuffer. The formulations may also comprise amino acids such as glycineand alanine and/or mannitol or other sugar alcohols and/or glyceroland/or other carbohydrates and optionally a preservative such as benzylalcohol.

International Patent Publication No. WO 94/03198 discloses a stableaqueous formulation containing human growth hormone, a buffer, anon-ionic surfactant, and, optionally, a neutral salt, mannitol, or, apreservative. The buffer may be histidine even though citrate ispreferred. The application discloses a preferred formulation containingcitrate as buffer, and natrium chloride and polysorbate 20 (Tween 20)for stabilization. This combination stabilizes the said aqueousformulation against aggregation but does give rise to a considerateamount of deamidated growth hormone.

International patent Publication No. WO 95/35116 discloses a stablelyophilized formulation containing saccharose and, optionally, mannitol.

DESCRIPTION OF THE INVENTION

It has now surprisingly been found that a pharmaceutical formulationcomprising growth hormone, or a derivative of growth hormone, and anamino acid selected from the group of Asp, Ile, Val or Leu, histidine,or a derivative of histidine, or or a peptide comprising at least onebasic amino acid residue and at least one acidic amino acid residue, anda non-ionic detergent, wherein the amount of peptide, or Asp, Ile, Val,Leu or His, or derivative of histidine is from about 0.01 to about 10 mgper mg of GH shows a high stability against deamidation and aggregation.The stability of the product allows for the storing and shipment thereofin a lyophilized state or in the form of a dissolved or re-dissolvedformulation.

The peptide comprising at least one basic amino acid residue and atleast one acidic amino acid residue to be used in accordance with thepresent invention may be a peptide comprising up to 20 amino acidresidues, preferably from 3 to 10 amino acid residues, more preferredfrom 3 to 6 amino acid residues such as 3 or 4 amino acid residues (e.g.Lys-Gly-Asp-Ser). In accordance with one aspect of the invention, thebasic and acid amino residues of shorter peptides are separated by 1 or2 amino acid residues. The peptides preferably comprise the naturallyoccurring alpha amino acid residues. The amino acid(s) may be 1 or damino acid(s) or a mixture thereof. “Acidic amino acid residues” aree.g. Glu or Asp, and “basic amino acid residues” are e.g. Lys or Arg.

The derivatives of histidine to be used in accordance with the presentinvention may be amides and esters of histidine such as the methyl orethyl ester, dipeptides having the sequence His-X or X-His, where X is anaturally occurring a-amino acid residue, and analogues or derivativesof His such as imidazole, des-amino-His or poly-His.

In a preferred aspect, the invention relates to a pharmaceuticalformulation comprising growth hormone (GH) or a derivative of growthhormone and an amino acid selected from the group of Asp, Ile, Val orLeu, and a non-ionic detergent, wherein the amount of the amino acidselected from the group of Asp, Ile, Val or Leu is from about 0.01 toabout 10 mg per mg of GH. In another preferred aspect, the inventionrelates to a pharmaceutical formulation comprising growth hormone (GH)or a derivative of growth hormone and histidine, or a derivative ofhistidine, or a peptide comprising at least one basic amino acid residueand at least one acidic amino acid residue, and a non-ionic detergent,wherein the amount of peptide or histidine or derivative of histidine isfrom about 0.01 to about 10 mg per mg of GH.

In a preferred embodiment of the above aspect, the amount of peptide, orselected amino acid, or histidine, or derivative of histidine, is fromabout 0.05 to about 5 mg per mg of GH, more preferred from about 0.05 toabout 1 mg per mg of GH, even more preferred from about 0.05 to about0.7 mg per mg of GH, and even more preferred from about 0.05 to about0.5 mg per mg of GH.

In a preferred embodiment of the above aspect, the non-ionic detergentis selected from a polysorbate or a poloxamer, e.g. polysorbate 20,poloxamer 188, or poloxamer 407 (e.g. Pluronic® F68, Lutrol 127, Tween20), most preferred poloxamer 188.

In another aspect, the invention relates to a pharmaceutical formulationof growth hormone, or a derivative of growth hormone, comprising anamino acid selected from the group of Asp, Ile, Val, Leu or His, or aderivative of histidine, or a peptide comprising at least one basicamino acid residue and at least one acidic amino acid residue, and anon-ionic detergent, wherein the amount of non-ionic detergent is fromabout 0.01 to about 10 mg per mg of GH.

In a preferred aspect, the invention relates to a pharmaceuticalformulation comprising growth hormone (GH) or a derivative of growthhormone, and an amino acid selected from the group of Asp, Ile, Val orLeu, and a non-ionic detergent, wherein the amount of non-ionicdetergent is from about 0.01 to about 10 mg per mg of GH. In anotherpreferred aspect, the invention relates to a pharmaceutical formulationcomprising growth hormone (GH), or a derivative of growth hormone, andhistidine, or a derivative of histidine, or a peptide comprising atleast one basic amino acid residue and at least one acidic amino acidresidue, and a non-ionic detergent, wherein the amount of non-ionicdetergent is from about 0.01 to about 10 mg per mg of GH.

In a preferred embodiment of the above aspect, the amount of non-ionicdetergent is from about 0.01 to about 10 mg per mg of GH, more preferredfrom about 0.05 to about 5 mg per mg og GH, even more preferred fromabout 0.1 to about 3 mg per mg of GH, even more preferred from about 0.1to about 2 mg per mg of GH.

In a preferred embodiment of the above aspect, the non-ionic detergentis selected from a polysorbate or a poloxamer, e.g. polysorbate 20,poloxamer 188, or poloxamer 407 (e.g. Pluronic® F68, Lutrol 127, Tween20), most preferred poloxamer 188.

Another aspect of the invention is a pharmaceutical formulationcomprising growth hormone, or a derivative of growth hormone, and anamino acid selected from the group of Asp, Ile, Val, Leu or His, or aderivative of histidine, or a peptide comprising at least one basicamino acid and at least one acidic amino acid, and a non-ionicdetergent, wherein the amount of peptide, or Asp, Ile, Val, Leu or His,or derivative of histidine, is from about 0.01 to about 10 mg per mg ofGH, and the amount of non-ionic detergent is from about 0.1 to about 2mg per mg of GH.

In a preferred aspect, the invention relates to a pharmaceuticalformulation comprising growth hormone or a derivative of growth hormone,and an amino acid selected from the group of Asp, Ile, Val or Leu, and anon-ionic detergent, wherein the amount of Asp, Ile, Val or Leu is fromabout 0.01 to about 10 mg per mg of GH, and the amount of non-ionicdetergent is from about 0.1 to about 2 mg per mg of GH. In anotherpreferred aspect, the invention relates to a pharmaceutical formulationcomprising growth hormone, or a derivative of growth hormone, andhistidine, or a derivative of histidine, or a peptide comprising atleast one basic amino acid residue and at least one acidic amino acidresidue, and a non-ionic detergent, wherein the amount of peptide orhistidine or derivative of histidine is from about 0.01 to about 10 mgper mg of GH, and the amount of non-ionic detergent is from about 0.1 toabout 2 mg per mg of GH.

In a preferred embodiment of the above aspect, the amount of peptide, orAsp, Ile, Val, Leu or His, or derivative of histidine, is from about0.05 to about 5 mg per mg of GH, more preferred from about 0.05 to about1 mg per mg of GH, even more preferred from about 0.05 to about 0.7 mgper mg of GH, and even more preferred from about 0.05 to about 0.5 mgper mg of GH.

In a preferred embodiment of the above aspect, the non-ionic detergentis selected from a polysorbate or a poloxamer, e.g. polysorbate 20,poloxamer 188, or poloxamer 407 (e.g. Pluronic® F68, Lutrol 127, Tween20), most preferred poloxamer 188.

Another aspect of the invention relates to a pharmaceutical formulationcomprising growth hormone, or an amino acid selected from the group ofAsp, Ile, Val, Leu or His, or a derivative of histidine, or a peptidecomprising at least one basic amino acid residue and at least one acidicamino acid residue, and a poloxamer.

In a preferred aspect, the invention relates to pharmaceuticalformulation comprising growth hormone (GH) or a derivative of growthhormone, and an amino acid selected from the group of Asp, Ile, Val orLeu, and a poloxamer. In another preferred aspect, the invention relatesto a pharmaceutical formulation comprising growth hormone, or aderivative of growth hormone, and histidine, or a derivative ofhistidine, or a peptide comprising at least one basic amino acid residueand at least one acidic amino acid residue, and a poloxamer.

In a preferrred embodiment of the above aspect, the poloxamer isselected from poloxamer 188, or poloxamer 407, most preferred poloxamer188 (e.g.Pluronic® F68). In another preferred embodiment, theformulation comprises histidine, or a derivative of histidine. In apreferred embodiment, the amount of peptide, or Asp, Ile, Val, Leu orHis, or derivative of histidine is from about 0.05 to about 5 mg per mgof GH, more preferred from about 0.05 to about 1 mg per mg of GH, evenmore preferred from about 0.05 to about 0.7 mg per mg of GH, and evenmore preferred from about 0.05 to about 0.5 mg per mg of GH. In apreferred embodiment, the amount of non-ionic detergent is from about0.05 to about 5 mg per mg of GH, more preferred from about 0.1 mg toabout 3 mg per mg of GH, more preferred from about 0.1 to about 2 mg permg of GH The formulation of the invention may be in the form of alyophilized powder to be reconstituted later using conventional vehiclessuch as distilled water, or water for injection, or it may be in theform of an aqueous solution comprising growth hormone. Such vehicles maycomprise conventional preservatives such as m-cresol, phenol, and benzylalcohol. As bulking agent(s) for lyophilization, one or more of themembers from the group consisting of sugar alcohols, e.g. mannitol, anddisaccharides, e.g. sucrose, may be selected.

Lyophilized preparations comprising sucrose are preferred due to a veryhigh stability, and preparations comprising sucrose and mannitol areespecially preferred combining very high stability with a very goodprocessability giving firm lyophilized products being readilydissolvable and very stable in solution for an extended period of timeafter dissolution. Further preferred preparations according to theinvention are preparations comprising mannitol and sucrose or trehaloseas bulking agent for the lyophilization. Preparations according to theinvention comprising mannitol and a disaccharide normally comprisesabout equal amount of the two constituents on a weight basis.

The amount of sucrose present in the preparations of the invention mayvary within wide limits. The ratio of growth hormone to sucrose may varyfrom 0.005 to 1.5 on a weight basis. Thus, the amount of sucrose may befrom 0.67 to 200 mg per mg of growth hormone, an amount of from 1.1 to50 mg per mg of growth hormone being preferred.

In a preferred embodiment of the invention, the growth hormone is humangrowth hormone (hGH) and the pharmaceutical composition is furthercomprising a carrier in the form of an aqueous buffer. Such an aqueouspreparation is in a ready-to-use form and may be stored and shipped assuch without any considerable degradation.

A buffer to be used in a solution of growth hormone may e.g. behistidine, citrate, tartrate, or phosphate buffer. L-histidine has a pKAof 6.0 and is, accordingly, suitable as a buffer itself in an intervalof pH from about 5 to about 7.

In a further aspect, the invention relates to a method of preparing apharmaceutical formulation comprising a growth hormone, or a derivativeof growth hormone, and an amino acid selected from the group of Asp,Ile, Val, Leu or His, or a derivative of histidine or a peptidecomprising at least one basic amino acid residue and at least one acidicamino acid residue, and a non-ionic detergent, wherein the appropriateamount of growth hormone, or derivative of growth hormone, is dissolvedin a solution comprising an amino acid selected from the group of Asp,Ile, Val, Leu or His, or a derivative of histidine, or a peptidecomprising at least one basic amino acid residue and at least one acidicamino acid residue in deionized water, and a non-ionic detergent, e.g. apolysorbate or a poloxamer, optionally containing a preservative, andoptionally containing an agent for adjusting the tonicity, andoptionally adjusting the pH to a value from about 6.0 to about 8.8; theappropriate amount of GH being what makes the amount of peptide, Asp,Ile, Val, Leu or His, or derivative of histidine range from about 0.01to about 10 mg per mg of GH.

In a further aspect, the invention relates to a method of preparing apharmaceutical formulation comprising a growth hormone, or a derivativeof growth hormone, and, an amino acid selected from the group of Asp,Ile, Val, Leu or His, or a derivative of histidine or a peptidecomprising at least one basic amino acid residue and at least one acidicamino acid residue, and a non-ionic detergent, wherein the growthhormone, or derivative of growth hormone, is dissolved in a solutioncomprising an amino acid selected from the group of Asp, Ile, Val, Leuor His, or a derivative of histidine or a peptide comprising at leastone basic amino acid residue and at least one acidic amino acid residuein deionized water, and a appropriate amount of non-ionic detergent,e.g. a polysorbate or a poloxamer, optionally containing a preservative,and optionally containing an agent for adjusting the tonicity, andoptionally adjusting the pH to a value from about 6.0 to about 8.8; theappropriate amount of non-ionic detergent being what makes the amount ofnon-ionic detergent range from about 0.01 to about 10 mg per mg of GH.In a further aspect, the invention relates to a method of preparing apharmaceutical formulation comprising a growth hormone, or a derivativeof growth hormone, and an amino acid selected from the group of Asp,Ile, Val, Leu or His, or a derivative of histidine, or or a peptidecomprising at least one basic amino acid residue and at least one acidicamino acid residue, and a non-ionic detergent, comprising dissolvingsuch an amount of growth hormone, or derivative of growth hormone, thatmakes the amount of peptide , or Asp, Ile, Val, Leu or His, orderivative of histidine range from about 0.05 to about 0.5 mg per mg GH,in a solution comprising an amino acid selected from the group of Asp,Ile, Val, Leu or His, or a derivative of histidine, or a peptidecomprising at least one basic amino acid residue and at least one acidicamino acid residue, and such an amount of a non-ionic detergent, e.g. apolysorbate or a poloxamer, that the amount of non-ionic detergent isfrom about 0.1 to about 2 mg per mg of GH, in deionized water,optionally containing a preservative, and optionally containing an agentfor adjusting the tonicity, and optionally adjusting the pH to a valuefrom about 6.0 to about 8.8.

In a further aspect, the invention relates to a method of preparing apharmaceutical formulation comprising a growth hormone, or a derivativeof growth hormone, and an amino acid selected from the group of Asp,Ile, Val, Leu or His, or a derivative of histidine, or a peptidecomprising at least one basic amino acid residue and at least one acidicamino acid residue, and a poloxamer, wherein the growth hormone, orderivative of growth hormone, is dissolved in a solution comprising anamino acid selected from the group of Asp, Ile, Val, Leu or His, or aderivative of histidine, or a peptide comprising at least one basicamino acid residue and at least one acidic amino acid residue indeionized water, and a poloxamer, optionally containing a preservative,and optionally containing an agent for adjusting the tonicity, andoptionally adjusting the pH to a value from about 6.0 to about 8.8.

The concentration of the amino acid selected from the group of Asp, Ile,Val, Leu or His, or a derivative of histidine, or a peptide comprisingat least one basic amino acid and at least on acidic amino acid ispreferably from about 1 mM to about 100 mM. More preferred, theconcentration of Asp, Ile, Val, Leu, or His, or derivative of histidine,or peptide comprising at least one basic amino acid and at least onacidic amino acid is from about 2 mM to about 20 mM, most preferred fromabout 5 mM to about 15 mM.

The pharmaceutical formulations of the invention may be formulated foradministration in any suitable way, e.g. by parenteral or oraladministration or administration to a mucosal membrane, e.g. nasaladministration. The pharmaceutical formulation may be presented in theform of a dose comprised in a vial or cartridge or any other suitablecontainer such as a prefilled syringe or a pen device.

The pharmaceutical preparation of the invention may furthermore comprisesalts conventionally used in order to facilitate the processing thereof,e.g. the lyophilization or reconstitution.

For stability reasons the pH of the solution is preferably adjusted to avalue in the interval from about 6.0 to about 8.8, preferably from about6.0 to about 7.0, more preferred from about 6.0 to about 6.8, even morepreferred from about 6.0 to 6.3, and most preferred from about 6.0 to6.2 (e.g. about 6.1).

The pH may be adjusted by adding an acid which has no adverse effect onthe growth hormone, preferably a physiologically acceptable acid e.g. amineral acid such as hydrochloric acid, sulphuric acid or nitric acid oran organic acid such as acetic acid or citric acid.

The pharmaceutical formulation of the invention may furthermore comprisesalts for adjusting the tonicity and optionally an excipient in order tofacilitate the processing thereof, e.g. lyophilization and the rapid andcomplete dissolution of a lyophilized formulation when reconstitutingthe formulation before use.

Such salts may be selected from conventional additives such as alkalinemetal, alkaline earth metal or ammonium salts of organic acids such ascitric acid, tartaric acid or acetic acid, e.g. sodium citrate, sodiumtartrate or sodium acetate, or of mineral acids such as hydrochloricacid, e.g. sodium chloride.

An excipient may be selected from disaccharides such as lactose,trehalose, and sucrose, sugar alcohols such as mannitol, xylitol,erythritol, threitol, sorbitol or glycerol, polysaccharides such as thepolymers commercialized as Dextran® products such as Dextran® 40,Dextran® 70 or Dextran® 75, and Ficoll® and polyvalent alcohols such aspolyethylene glycol or polyvinyl alcohol or a combination of two or moreof these.

In the present context “growth hormone” or “GH” may be growth hormonefrom any origin such as avian, bovine, equine, human, ovine, porcine,salmon, trout or tuna growth hormone, preferably bovine, human orporcine growth hormone, human growth hormone being most preferred. Thegrowth hormone used in accordance with the invention may be nativegrowth hormone isolated from a natural source, e.g. by extractingpituitary glands in a conventional manner, or a growth hormone producedby recombinant techniques, e.g as described in E. B. Jensen and S.Carlsen in Biotech and Bioeng. 36, 1-11 (1990). The “growth hormonederivative” may be a truncated form of growth hormone wherein one ormore amino acid residues has (have) been deleted; an analogue thereofwherein one or more amino acid residues in the native molecule has(have) been substituted by another amino acid residue, preferably theresidue of a naturally occurring amino acid, as long as the substitutiondoes not have any adverse effect such as antigenicity or reduced action;or a derivative thereof, e.g deamidated or sulfoxidated forms of thegrowth hormone or forms having an N- or C-terminal extension such asMet-hGH, Met-Glu-Ala-Glu-hGH or Ala-Glu-hGH. The preferred growthhormone is hGH. The growth hormone may be a concentrate obtaineddirectly from the fermentation broth or a conventional lyophilizedpreparation which is dissolved in an appropriate solvent.

The term “derivatives of histidine” is used, for the present purpose, todesignate amides and esters of histidine such as the methyl or ethylester, dipeptides such as His-Gly, His-Ala, His-Leu, His-Lys, His-Ser,His-Phe, and dipeptides such as Ala-His, Gly-His, Val-His, Glu-His,Met-His, Arg-His, Asp-His, Leu-His, Ser-His, and analogues orderivatives of His such as imidazole, des-amino-His or poly-His.

In the present context “non-ionic detergent” or “non-ionic surfactant”may be polysorbates, such as polysorbate 20 or 80, etc., and thepoloxamers, such as poloxamer 188 or 407, Pluronic® polyols, and otherethylene/polypropylene polymers, etc. Amounts effective to provide astable, aqueous formulation will be used, usually in the range of fromabout 0.01 to about 10 mg detergent per mg of growth hormone, preferablyfrom about 0.05 to about 5 mg detergent per mg of growth hormone, morepreferred from about 0.1 to about 2 mg detergent per mg of growthhormone.

The use of non-ionic detergents permits the formulation to be exposed toshear and surface stresses without causing denaturation of the protein.

The amount of non-ionic detergent in the formulations may be in therange of from 0.01 to about 10 mg detergent per mg of growth hormone,preferably from 0.05 to about 5 mg detergent per mg of growth hormone,most preferred from 0.1 to about 2 mg detergent per mg of growthhormone.

In the present context “disaccharide” is used to designate naturallyoccurring disaccharides such as sucrose, trehalose, maltose, lactose,sepharose, turanose, laminaribiose, isomaltose, gentiobiose ormelibiose.

A peptide comprising at least one basic amino acid residue and at leastone acidic amino acid residue to be used in accordance with the presentinvention is preferably comprising the naturally occurring α-amino acidresidues. The amino acid(s) may be 1 or d amino acid(s) or a mixturethereof A preferred peptide comprising at least one basic amino acidresidue and at least one acidic amino acid residue is Lys-Gly-Asp-Ser.

In the present context “acidic amino acid residues” are e.g. Glu or Asp,and “basic amino acid residues” are e.g. Lys or Arg.

The solvent used in the method of the invention may be water, alcoholssuch as ethyl, n-propyl or isopropyl, butyl alcohol or mixtures thereof.To retard microbial growth, the solvent may comprise a preservative suchas m-cresol, phenol, or benzyl alcohol.

The term “dose” of growth hormone refers to that amount that providestherapeutic effect in an administration regimen. The formulations hereofare prepared containing amounts of hGH at least about 0.1 mg/ml,preferably upwards of about 1 mg/ml, preferably from about 1 mg/ml toabout 40 mg/ml, more preferably from about 1 mg/ml to about 25 mg/ml,more preferably from about 1 mg/ml to about 15 mg/ml e.g. from 1 mg/mlto about 10 mg/ml, calculated on the ready-to-use formulation. For useof these compositions in administration to human beings suffering fromhypopituitary dwarfism, for example, these formulations contain fromabout 0.1 mg/ml to about 10 mg/ml, corresponding to the currentlycontemplated dosage regimen for the intended treatment. Theconcentration range is not critical to the invention and may be variedby the physician supervising the administration.

Another aspect of the invention relates to a method of treating apatient affectable by growth hormone comprising treating the patientwith an amount of the pharmaceutical formulation according to theinvention effective to treat said disorder.

The invention is explained more in detail in the below Examples whichillustrate the invention. They are not to be considered as limiting thescope of the invention being defined by the appended claims.

EXPERIMENTAL PART cl EXAMPLE 1

Inhibition of Aggregation

Liquid formulations of hGH was prepared by dissolving hGH in a solutionof excipients and adjusting the pH with HCl/NaOH. The composition of theformulations were: hGH 5 mg/ml Mannitol 45 mg/ml L-histidin 0.62 mg/mlPhenol 2.5 mg/ml Surfactant x mg/mlpH = y (from 6.1 to 6.8)

The appearance of the solutions was examined visually after thesolutions had been stored 1 day at 8° C. and rotated for 19 hour (20rpm) at 25° C., respectively: x pH Appearance after Surfactant (mg/ml)(y) 1 day at 8° C. Appearance after 19 hours rotation- 0 6.1Precipitation Precipitation — 0 6.3 Precipitation Many flakes — 0 6.5Flakes Many flakes — 0 6.8 Flakes Some flakes Pluronic L64 0.5 6.8Clear, few flakes Many small flakes Pluronic L64 1 6.8 Clear, few flakesClear, some flakes Pluronic F68 2 6.1 Clear Clear, some flakes PluronicF68 2 6.3 Clear Clear, few flakes Pluronic F68 2 6.5 Clear Clear, fewflakes Pluronic F68 2 6.8 Clear Clear, some flakes Lutrol F127 1 6.1Clear Clear, some flakes Lutrol F127 1 6.3 Clear Clear, some flakesLutrol F127 1 6.5 Clear Clear, few flakes Lutrol F127 1 6.8 Clear Notanalysed Tween 20 2 6.1 Slight Reasonably clear, precipitation someflakes Tween 20 2 6.3 Clear Clear, some flakes Tween 20 2 6.5 Clear,some flakes Clear, few flakes Tween 20 2 6.8 Clear Clear, some flakes

EXAMPLE 2

Rate of Deamidation

The eight formulations tabulated below were prepared by adding the hGHto a solution containing the excipients. pH was adjusted to 6.1. Thepreparations were then placed for 28 days at 25° C. and for 14 days at37° C. and analysed for formation of deamidated forms of hGH. Rateconstants for the deamidation were calculated on basis of the stabilitydata. In the table below the contents of hGH and excipients are given inmg/ml and the rate constants k_(25° C.) and k_(37° C.) are given indays⁻¹ Disodium Pluronic Lutrol L- hydrogen hGH Phenol F68 F127 histidincitrate Mannitol NaCl k_(25° C.) k_(37° C.) 5 2.5 2 0.62 45 0.001800.00860 6.7 2.5 2 2.2 45 0.00221 0.0127 6.7 2.5 2 0.62 5.8 0.002440.0110 6.7 2.5 2 2.2 5.8 0.00263 0.0143 5 2.5 1 0.62 45 0.00178 0.009146.7 2.5 1 2.2 45 0.00221 0.0128 6.7 2.5 1 0.62 5.8 0.00236 0.0112 6.72.5 1 2.2 5.8 0.00259 0.013 9

EXAMPLE 3

Formation of Deamidated Forms

The formulations tabulated below were prepared by adding the hGH to asolution containing the excipients and adjusting the pH. Thepreparations were then placed for 3 months at 8° C. and 25° C. andanalysed for formation of deamidated forms of hGH. Content of desamidoforms (%) 3 weeks hGH Surfactant¹⁾ Phenol L-histidin Mannitol 3 months12 weeks at (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) pH t = 0 at 8° C. at25° C. 37° C. 6.25 2 (F68) 2.5 0.62 45 6.3 0.6 2.7 18.1 21.1 6.25 2(F68) 2.5 0.62 45 6.2 0.5 2.5 16.2 18.7 6.25 2 (F68) 2.5 0.62 45 6.1 0.62.2 14.8 17.1 3.33 3 (F68) 3.0 0.68 40 6.1 1.3 3.0 15.5 n.a. 6.67 3(F68) 3.0 0.68 40 6.1 1.0 3.2 16.6 n.a. 10.0 3 (F68) 3.0 1.1 39 6.1 0.63.8 16.7 n.a. 6.25 1 (L127)  2.5 0.62 45 6.1 0.6 2.5 15.4 18.2 6.25 2(T20) 2.5 0.62 45 6.1 0.6 n.a. n.a. 16.6¹⁾Type of surfactant is given in parantheses:F68 = Pluronic F68 = poloxamer 188F127 = Lutrol 127 = poloxamer 407T20 = Tween 20 = polysorbate 20

EXAMPLE 4

24 Formulations were Prepared from the Below Formula:

-   hGH3.33 to 13.1 mg/ml-   L-histidin 0.78 mg/ml-   Mannitol22 mg/ml-   Sucrose21 mg/ml-   Poloxamer 1881.33-5.33 mg/ml-   Preservative: Benzylalcohol (0-20 mg/ml) or Phenol (0-5 mg/ml)

The formulations were prepared by adding the hGH to a solution of theexcipients. pH was adjusted to 6.8. The formulations were kept at 25° C.for 4 weeks and then the clarity of the solutions were measured as theabsorbance at 340 nm. The absorbance at 340 nm indicate the degree ofaggregation in the solutions. Benzyl- alcohol Phenol (mg/ml) (mg/ml) hGHPoloxamer (mg/ml) 188 (mg/ml) 0 15 18 20 2.5 5 3.33 1.33 0.009 0.0190.045 0.016 0.015 0.020 6.67 2.66 0.046 0.022 0.041 1) 0.025 0.026 10.04.0 0.022 0.060 1) 1) 0.034 0.050 13.1 5.33 0.018 0.041 0.042 1) 0.0540.0711): dissolution of hGH not possible.

REFERENCES

-   1) Y.-C. J. Wang and M. A. Hanson. Parenteral Formulations of    Proteins and Peptides: Stability and Stabilizers. J. Parenteral    Science and Technology 42 (Suppl.) (1988) 53-525.-   2) M. C. Manning, K. Patel, R. T. Borchardt. Stability of Protein    Pharmaceuticals. Pharmaceutical Research 6 (11) (1989) 903-918.-   3) B. A. Johnson, J. M. Shirokawa, W. S. Hancock, M. W.    Spellman, L. J. Basa and D. W. Asward. J. Biol. Chem. 264, 1462-71    (1989).-   4) L. C. Teh et al., J. Biol. Chem., 262, 785-794 (1987).-   5) G. W. Becker et al., Biotech. Appl. Biochem., 10, 326-337 (1988).-   6) R. A. Houghten et al., Arch. Biochem. Biophys., 178, 350-355    (1977).-   7) R. M. Riggin et al., Anal. Biochem., 167, 199-209 (1987).-   8) P. Gellerfors et al., Acta Paediatr. Scand (suppl), 370, 93-100    (1990).-   9) M. J. Kaufman, Pharm. Res., 7 (3) 289-292 (1990).

1. An aqueous pharmaceutical formulation comprising a) about 1 mg/ml toabout 15 mg/ml of human growth hormone (hGH), b) histidine in an amountof about 0.05-about 0.5 mg per mg hGH, c) Poloxamer 188 in an amount ofabout 0.1-about 2 mg per mg hGH, d) mannitol in an isotonicityconferring amount, and e) phenol in an amount that confers apreservative effect, wherein the formulation has a pH of about 6.0-about6.3.
 2. The formulation of claim 1, wherein the formulation has a pH ofabout 6.1.